Skeletal muscle exemplifies structure-function relationships in biology. The organization of sarcomeres follow hierarchical ordering to form long contractile cells, bundled in extra-cellular matrix, to form larger fascicles and ultimately whole muscles. The tight relationship between structure and function allows muscle performance (and disease) to be inferred from its microstructure. For example, fiber area is directly related to isometric force production in muscle. With injury, microstructural changes in muscle fiber area (size), fibrosis (accumulation of extracellular matrix), membrane damage (permeability), and inflammation (edema) are observed, and impair muscle function. Muscle biopsy, followed by microscopic examination of the tissue (histology), is the gold standard to diagnose and monitor muscle health and disease. This tool is invasive, requiring a large bore needle and tissue removal under sterile conditions, which makes it painful and costly. Therefore, biopsy is not conducive to serial monitoring of muscle health. It is also semi-quantitative, and often difficult to extrapolate to the entire muscle, limiting its scientific and clinical value. For these reasons, there is a need for noninvasive assessment of muscle microstructure, which would facilitate the quantitative examination of muscle injury over time. Magnetic resonance imaging (MRI) has been used to noninvasively quantify changes in volume, fat distribution, and water content in muscle. Diffusion tensor imaging (DTI) is a version of MRI that measures anisotropic diffusion of water, which is related to tissue microstructure, but tends to yield non-specific changes regardless of the injury or disease state. The key reason for this lack of specificity is that the explicit relationships between microstructure and diffusion have not been rigorously studied, nor carefully calibrated. To address this gap in knowledge, the purpose of this proposal is to compare muscle microstructure and MRI diffusion properties of muscle in novel and tightly controlled computer simulations, precision engineered phantoms, and animal models of muscle injury and disease. Our central hypothesis is that DT-MRI can be directly related to muscle microstructural changes, when appropriate pulse sequences are used to uncouple complex pathology. Aim #1 will use computer-based simulations of muscle structure and biochemistry to carefully understand how diffusion is related to multiple muscle microstructural changes. Aim #2 will utilize 3D precision-engineered models to relate diffusion to muscle structure in real-world DT-MRI experiments. These experiments will be integrated into a final in vivo set of experiments (Aim #3), which are designed to test the accuracy of DT-MRI to uncouple complex microstructural changes in the presence of muscle atrophy, inflammation, and degeneration. These experiments will elucidate the understudied relationships between microstructure and diffusion in muscle. The long-term goal is to serially quantify muscle microstructure non- invasively. This approach is innovative in that it combines state-of-the art imaging, simulation, nanofabrication, and morphology methods to generate a clinically meaningful measurement tool.